Method for culturing primary cells (PDC) from glioma patient

Abstract

The invention relates to the related technical field of primary cell (PDC) culture, in particular to a method for culturing primary cells (PDC) from glioma patients, which comprises a series of steps of Swis3T3-J2 cell culture, conditioned medium preparation, brain tumor tissue pretreatment, primary tumor cell digestion and culture and the like. Primary human tumor cells are cultured by a conditioned culture medium generated by fibroblasts irradiated by X-rays (X) and a cobalt-60 radioactive source (60Co), and culture conditions of key features of parent tumors and a modeling process can be most effectively reserved by screening and optimizing the culture conditions. And whether the PDC retains the key characteristics of the parent tumor or not is inspected on the aspects of the cell molecular level, the cell phenotype, the animal intracranial tumor model and the like.

Description

Technical Field

[0001] The present invention relates to the technical field related to primary cell (PDC) culture, and in particular to a method for culturing primary cells (PDC) derived from glioma patients. Background Art

[0002] The existing glioma cell lines are difficult to truly reflect the patient's tumor characteristics, mainly reflected in the inability to retain the tumor's transcriptional characteristics, gene mutations, and intratumor heterogeneity. Immortalized cell lines limit researchers' in-depth understanding of the occurrence and development of gliomas and the development of effective treatment strategies. Therefore, it is particularly important to construct a primary cell model that can better represent the tumor characteristics of glioma patients and develop an in situ animal model based on primary cells. Primary cells and animal models can theoretically more accurately simulate patients' tumors, including tumor heterogeneity and microenvironment, and can provide powerful tools for studying the pathogenesis of gliomas, drug targets, and developing new therapies.

[0003] The existing methods for culturing primary glioma cells mainly focus on the cultivation of tumor stem cells, including monolayer culture and tumor spheroid culture; however, traditional culture methods require a lot of time to establish, and the exogenous EGF and bFGF used selectively amplify tumor stem cells and cannot retain other tumor cell subpopulations. Patient-derived tumor tissue xenograft models (PDX) can better simulate the intratumor heterogeneity of tumor tissue, but their transplantation efficiency is low, and the tumor growth time ranges from 2 to 11 months, which is not suitable for clinical personalized treatment testing. In recent years, glioma organoid models (PDO) have gradually developed. PDO models can well show the heterogeneity between and within tumors. However, the materials such as matrix gel used in this type of organ culture are expensive, and the cell passaging process is relatively complicated. During high-throughput drug screening, PDO cannot be quickly and evenly planted in 384 or 1536-well plates, which is not suitable for large-scale drug trials. Therefore, it is crucial to develop an effective primary glioma cell model suitable for high-throughput drug screening. Summary of the invention

[0004] The purpose of the present invention is to provide a method for culturing primary cells (PDC) derived from glioma patients. In order to overcome the problem that the cell passage process is relatively complicated in the technical background, PDO cannot be quickly and evenly planted in 384 or 1536-well plates during high-throughput drug screening, and is not suitable for large-scale drug trials, the present invention proposes a method for culturing primary cells (PDC) derived from glioma patients to alleviate the aforementioned problems.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for culturing primary cells (PDC) derived from glioma patients, the specific steps of which are as follows:

[0006] S1, Swiss3T3-J2 cell culture:

[0007] S11 and Swiss3T3-J2 mouse fibroblasts were cultured in DMEM complete medium and cell passage was performed after the cell confluence rate reached 60%-70%;

[0008] When subculturing S12 and J2 cells, the culture medium was discarded, the adherent cells were washed once with PBS, and digested with trypsin / EDTA solution;

[0009] S13. Observe the cell digestion status under a microscope. When the cells become round and begin to fall off from the bottom of the culture dish, gently tap the culture bottle to promote cell shedding.

[0010] S14. Stop the trypsin / EDTA action by adding four volumes of DMEM complete medium to the cells;

[0011] S15, centrifuge at 300g for 5 minutes at 4°C, resuspend the cells in 10 mL of DMEM complete medium, and inoculate 3 × 10 cells per square centimeter. 3 Cells, passaged in about three days;

[0012] S2. Conditioned medium preparation:

[0013] S21, Swiss3T3-J2 cells were suspended in DMEM complete medium, and irradiated with 6MV electron beam, X-ray and cobalt-60 radiation sources at radiation doses of 10 Gy, 30 Gy and 60 Gy, respectively;

[0014] S22, after irradiation, 1.0×10 7 to 1.5×10 7 The cells were seeded at 175 cm 2 Add 30 mL of F medium to the tissue culture flask;

[0015] S23, 72 h later, the culture medium produced by 3T3 cells was collected and centrifuged at 1000 g for 5 min at 4 °C;

[0016] S24, filtering the culture medium with a filter, freezing the treated culture medium at -80°C, and before use, mixing three portions of the culture medium produced by 3T3 cells with one portion of fresh F culture medium, and adding Y-27632 (10 μM) to prepare conditioned medium CM;

[0017] S3. Pretreatment of brain tumor tissue:

[0018] S31. Within 1 hour after surgery, quickly rinse the excised tissue sample with 4°C precooled PBS, transfer the tissue to a sterile culture dish, remove the remaining fat tissue with forceps and a scalpel, and cut into approximately 1 cm3 The tumor was cut out as completely as possible from the surrounding normal tissue, and at least one section of each sample was used for pathological examination;

[0019] S32, place the sample in F medium (containing 10 μM Y-27632) and transport at 4°C;

[0020] S4. Digestion and culture of primary tumor cells:

[0021] S41, dilute 10× collagenase / hyaluronidase solution to 1× with F medium, mix 3 mL of F medium / collagenase / hyaluronidase with 1 mL of dispase;

[0022] S42. Rapidly rinse the excised tissue sample with ethanol and then rinse with ice-cold PBS;

[0023] S43, take 1cm 3 Use a scalpel to mince the tissue.

[0024] S44. Transfer tissue fragments to a tube containing 4 mL of F medium / collagenase / hyaluronidase / dispase and incubate on a shaker for digestion.

[0025] S45. After the tissue is fully enzymatically separated, centrifuge at 500 g for 5 min at 4°C, discard the supernatant, and resuspend the cells in 10 mL of F medium;

[0026] S46. Filter the cell suspension through a 100 μm cell sieve into a new 50 mL centrifuge tube;

[0027] S47. Divide the cell suspension equally according to the type of culture medium and add it to the centrifuge tube. Centrifuge at 300g for 5 minutes at 4°C. Discard the supernatant to collect the cell pellet. Resuspend the cells with CM conditioned medium and then plant the cells in a T75 cell culture flask.

[0028] Preferably, the J2 cells in the S11 are prone to growth inhibition, and the growth density during subculture cannot exceed 4×10 4 / cm 2 .

[0029] Preferably, the condition for washing the adherent cells once with PBS in S12 is digesting them with 0.05% trypsin / EDTA solution at room temperature for 1 minute.

[0030] Preferably, the F culture medium formula in S22 (volume or final concentration in brackets) is: DMEM complete culture medium (373 mL), F12 nutrient mix (125 mL), Hydrocortisone (25 ng / mL), EGF (0.125 ng / mL), Insulin (5 μg / mL), cholera toxin (0.1 nM), Amphotericin B (250 ng / mL), and gentamicin (10 mg / mL).

[0031] Preferably, the filter in S24 is a Millex-GP filter with a pore size of 0.22 μm.

[0032] Preferably, the concentration of ethanol in S42 is 95%-100%, and the flushing time is at most 3 seconds.

[0033] Preferably, the incubation digestion temperature on the shaker in S44 is 37° C. and the time is 1-3 hours.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] Using high energy electrons (E), X-rays (X) and cobalt-60 radiation sources ( 60 Primary human tumor cells were cultured with the conditioned medium produced by Co)-irradiated fibroblasts. By screening and optimizing the culture conditions, the culture conditions and modeling processes that most effectively retain the key characteristics of the parental tumor were identified. The key characteristics of the parental tumor were examined at the cellular molecular level, cell phenotype, and animal intracranial tumor model.

[0036] The conditional reprogramming technology was applied to the culture of primary glioma cells. Compared with glioma cell lines, the constructed PDCs performed well in simulating the heterogeneity of patient tumors. The PDCs cultured in vitro showed molecular characteristics of brain neurons and expressed glioma molecular markers such as Nestin, CD44, O4 and GFAP. RNA sequencing of PDCs showed that they had extremely high similarity with the parental tumor at the transcriptional level. Whole exome sequencing showed that PDCs retained the DNA variation characteristics of tumor cells and were consistent with the parental tumor in the typical glioma mutation IDH1R132H. PDCs can be efficiently implanted into the brains of adult mice to form tumors. The animal model also showed typical glioma histological characteristics. The tumor tissue was highly malignant, with GBM tumor characteristics such as cortical invasion and infiltration, distal migration and neuronal encirclement. Characterization based on the molecular and biological phenotypes of PDCs showed that the constructed PDCs can well simulate the in vivo state of tumors.

[0037] Compared with the traditional glioma stem cell culture method, the conditional reprogramming method used greatly retains the intratumoral cell heterogeneity. Single-cell sequencing of PDCs showed that tumor cells included multiple cell populations existing in tumors, such as MES-like, astroglioma cell-like, OPC-like cells and stem cells. Analysis based on single-cell CNV showed that PDCs retained the abnormal chromosomal characteristics of tumors, such as the common amplification of chromosomes 7 and 8 and the loss of chromosomes 6, 10, 13 and 14 in GBM, while the stem cell culture method could not retain the chromosomal variation of tumor cells. Amplification of chromosome 7 is a common chromosomal variation in gliomas, which includes the region encoding EGFR. The high expression of EGFR in gliomas is associated with this chromosomal change, while amplification of chromosome 8 can lead to the overexpression of many oncogenes, such as MYC. The loss of the chromosome 10 region often involves the loss of PTEN. As an important tumor suppressor gene, the loss of PTEN is associated with the high invasiveness and poor prognosis of the tumor. Chromosome variation is an important factor in the occurrence and development of gliomas. The constructed PDC retains the characteristics of the patient in terms of chromosome variation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The figure is a schematic diagram of the process of culturing primary cells (PDC) derived from glioma patients of the present invention;

[0039] Figure 2 This is a schematic diagram of screening feeder cell irradiation conditions in the conditional reprogramming of the primary cell (PDC) culture method derived from glioma patients of the present invention;

[0040] Figure 3 The schematic diagram of the glioma patient-derived primary cell (PDC) culture method of the present invention and the glioma PDC and traditional stem cell culture;

[0041] Figure 4 Schematic diagram of the glioma patient-derived primary cell (PDC) culture method of the present invention, showing that 5 PDCs cultured with E-30Gy CM express glioma patient glial cell, precursor cell and stem cell markers;

[0042] Figure 5 A schematic diagram of the mRNA expression similarity between 10 PDCs and matched tumors in the culturing method of primary cells (PDCs) derived from glioma patients of the present invention;

[0043] Figure 6 A schematic diagram of single cell clustering analysis of the primary cell (PDC) culture method derived from glioma patients of the present invention;

[0044] Figure 7 Schematic diagram of the primary cell (PDC) culture method from glioma patients of the present invention, tumor samples and PDC mutation detection and analysis;

[0045] Figure 8 This is a schematic diagram of the construction of the PDC in situ glioma mouse (PDC_DX) for the primary cell (PDC) culture method derived from glioma patients of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] This embodiment discloses a method for culturing primary cells (PDC) derived from glioma patients, such as Figure 1-8 As shown, the specific steps are as follows:

[0048] S1, Swiss3T3-J2 cell culture:

[0049] Swiss3T3-J2 mouse fibroblasts were cultured in DMEM complete medium. Cells were passaged after the cell confluence reached 60%-70%. When J2 cells were passaged, the medium was discarded first, the adherent cells were washed once with PBS, and then digested with trypsin / EDTA solution. The cell digestion status was observed under a microscope. When the cells were round and began to fall off from the bottom of the culture dish, the culture flask was gently tapped to promote cell shedding. The effect of trypsin / EDTA was terminated by adding four times the volume of DMEM complete medium to the cells. S15, centrifuged at 300g for 5 minutes at 4°C, resuspended the cells in 10mL DMEM complete medium, and 3×10 3 Cells were passaged in about three days.

[0050] S2. Conditioned medium preparation:

[0051] Swiss3T3-J2 cells were suspended in DMEM complete medium and irradiated with 6MV electron beam, X-ray and cobalt-60 radiation sources at 10 Gy, 30 Gy and 60 Gy, respectively. After irradiation, 1.0×10 7 to 1.5×10 7 The cells were seeded at 175 cm 230 mL of F medium was added to a tissue culture flask. After 72 hours, the medium produced by 3T3 cells was collected and centrifuged at 1000 g for 5 minutes at 4°C. The medium was filtered using a filter and the treated medium was frozen at -80°C. Before use, three portions of the medium produced by 3T3 cells were mixed with one portion of fresh F medium, and Y-27632 (10 μM) was added to prepare the conditioned medium CM.

[0052] Conditioned medium produced by irradiated mouse fibroblasts and the Rho-associated kinase (ROCK) inhibitor Y-27632 were used as the main culture conditions. Y-27632 was able to increase the cloning efficiency of human embryonic stem cells and the survival rate of human keratinocyte stem cells. Normal cells cultured using conditional reprogramming technology maintained their developmental potential and could differentiate into the normal tissues from which they were derived when re-transplanted back into the body. Tumor cells derived from peripheral organs cultured using conditional reprogramming technology maintained the phenotypic and genotypic characteristics of the parental tumor.

[0053] S3. Pretreatment of brain tumor tissue:

[0054] Within 1 hour after surgery, quickly rinse the excised tissue sample with 4°C precooled PBS, transfer the tissue to a sterile culture dish, remove the remaining fat tissue with forceps and a scalpel, and cut into approximately 1 cm 3 The diseased tumor was cut out as thoroughly as possible from the surrounding normal tissue in small pieces. At least one section of each sample was used for pathological examination. The samples were placed in F medium (containing 10 μM Y-27632) and transported at 4°C.

[0055] S4. Digestion and culture of primary tumor cells:

[0056] Dilute 10× collagenase / hyaluronidase solution to 1× with F medium, mix 3 mL F medium / collagenase / hyaluronidase with 1 mL dispase, quickly rinse the excised tissue sample with ethanol, then rinse with ice-cold PBS, and take 1 cm 3 The tissue blocks were minced with a scalpel, and the tissue fragments were transferred to a test tube containing 4 mL of F medium / collagenase / hyaluronidase / dispase. The tissues were incubated and digested on a shaker. After the tissues were fully enzymatically hydrolyzed and separated, they were centrifuged at 500 g for 5 minutes at 4°C, the supernatant was discarded, the cells were resuspended in 10 mL of F medium, and the cell suspension was filtered through a 100 μm cell sieve into a new 50 mL centrifuge tube. The cell suspension was evenly divided according to the type of culture medium and added to the centrifuge tubes. The cells were centrifuged at 300 g for 5 minutes at 4°C, the supernatant was discarded, the cell pellets were collected, the cells were resuspended with CM conditioned medium, and the cells were seeded in T75 cell culture flasks.

[0057] Furthermore, J2 cells in S11 are prone to growth inhibition, and the growth density during subculture should not exceed 4×10 4 / cm 2 .

[0058] Furthermore, the conditions for washing the adherent cells once with PBS in S12 were followed by digestion with 0.05% trypsin / EDTA solution at room temperature for 1 minute.

[0059] Furthermore, the formula of F medium in S22 (volume or final concentration in brackets) is: DMEM complete medium (373 mL), F12 nutrient mix (125 mL), Hydrocortisone (25 ng / mL), EGF (0.125 ng / mL), Insulin (5 μg / mL), cholera toxin (0.1 nM), Amphotericin B (250 ng / mL), and gentamicin (10 mg / mL).

[0060] Furthermore, the filter in S24 is a Millex-GP filter with a pore size of 0.22 μm.

[0061] Furthermore, the concentration of ethanol in S42 is 95%-100%, and the flushing time is at most 3 seconds.

[0062] Furthermore, the incubation digestion temperature on the shaker in S44 is 37° C. and the time is 1-3 hours.

[0063] Embodiment 1: Figure 2 The radiation effects caused by different radiation sources and doses shown in A may be different. First, high-energy electrons (E), X-rays (X) and cobalt-60 radiation sources ( 60 The irradiation effect of Co) on feeder cells was studied to inhibit the proliferation of feeder cells and cause them to produce conditioned medium. By drawing the cell growth curve, it was found that the 3T3 cells in the control group (non-irradiated group) proliferated rapidly, the growth curve of the 3T3 cells in the 10Gy dose group tended to be stable, the total number of cells in the 30Gy dose group decreased slightly, and the 3T3 feeder cells at a dose of 60Gy all died on the 8th day.

[0064] like Figure 2 As shown in Figure B, 3T3 feeder cells were irradiated with 10Gy and 30Gy doses from different radiation sources to prepare conditioned medium (CM). The growth of PDC in different CMs was observed under a microscope. PDC could quickly adhere to the wall and proliferate. There was no significant difference in morphology between the 10Gy and 30Gy CMs from the three radiation sources. PDC in E-CM and X-CM was larger than that in 60 The growth rate in Co-CM was slightly faster.

[0065] Figure 2 The growth curve of feeder cell 3T3-J2 after being treated with different irradiation sources and irradiation doses is shown in A. Figure 2 B shows the bright field observation of the cell morphology of PDC in different conditioned medium CM, cell generation P3. E: 6MeV high energy electrons; X: X-rays; 60 Co: Cobalt-60.

[0066] Example 2: By comparing the growth of primary cells from patients in CM and traditional glioma stem cell culture medium, it was found that the tumor cells in the stem cell culture medium grew extremely slowly. Although cytokines were added or fresh culture medium was replaced every 3 days, it still took 3-4 weeks to pass the culture once (e.g. Figure 3 The subculture period of PDC grown in CM is about 5-7 days (as shown in Figure A). Figure 3 As shown in B), cell counting revealed that the total number of cells in the formed 3D neurosphere did not increase. Under the microscope, a dark opaque area was observed in the center, which may be due to the presence of an oxygen-deficient area in the center of the neurosphere, causing some cells to die.

[0067] like Figure 3 As shown in A, during stem cell culture, the cell subculture cycle is about 3-4 weeks, and cytokines are added or fresh culture medium is replaced every 3 days. Figure 3 As shown in B, using CM culture, the PDC subculture cycle is about 7 days, SC: Stem cell.

[0068] Embodiment 3: Figure 4 As shown, immunofluorescence experiments were performed on 5 PDCs numbered YG027_C, YG038_C, YG043_C, YG046_C and YG056_C constructed with E-30Gy CM to detect their expression of neuronal cell-related markers, including glial cell markers GFAP, S100β and O4, oligodendrocyte precursor cell markers PDGFRα, NG2 and glioma stem cell markers NESTIN, CD44. The morphology and molecular expression of PDCs in different patients, for example: YG027_C highly expressed oligodendrocyte precursor cell and stem cell markers, YG038_C highly expressed oligodendrocyte markers and proliferation markers, YG043_C and YG056_C highly expressed stem cell and precursor cell markers. Immunofluorescence showed that PDCs exhibited obvious inter-tumor heterogeneity.

[0069] Embodiment 4: Figure 5As shown in Figure 2, by matching PDC and tumor tissues of 10 patients for RNA sequencing, the similarity between PDC and parental tumor at the cellular transcription level was compared, and low-expression transcripts with TPM values ​​less than 0.1 were filtered out. A total of 11,716 gene transcripts were obtained with Spearman correlation coefficients (e.g. Figure 5 At different abundances of cellular transcripts, such as 685 gene transcripts with RNA expression TPM greater than 40, 2845 transcripts with RNA expression TPM greater than 10, 10059 transcripts with RNA expression TPM greater than 1, and 11716 transcripts with RNA expression TPM greater than 0.1, PDC was strongly correlated with the parental tumor at these different thresholds of transcripts (as shown in Figure 2A). Figure 5 (shown in B).

[0070] Figure 5 As shown in A, the expression TPM value was calculated based on bam reads, and low-expression genes with TPM less than 0.1 were removed to obtain the spearman correlation coefficient between PDC and 11716 genes in tissues. Figure 5 As shown in B, based on different thresholds set by TPM, the heat map shows the spearman correlation between tissue and PDC. The paired samples of each patient are located on the diagonal line, and the values ​​are all greater than 0.75.

[0071] Furthermore, the ability of PDC to maintain intratumoral cell heterogeneity was evaluated by single-cell sequencing of three samples: YG021_T, the tumor tissue of YG021 patient, YG021_C cultured in reprogrammed CM, and YG021_SC cultured in traditional glioma stem cell culture medium. UMAP dimensionality reduction clustering analysis showed that conditionally reprogrammed YG021_C maintained almost all cell types in the original tumor tissue (except T cells and monocytes), including astrocytes, mesenchymal-like glioma cells, endothelial cells, and microglia / macrophages, while traditional stem cell culture methods mainly enriched neural stem cells and retained very few mesenchymal-like cells (such as Figure 6 As shown), wherein single-cell sequencing was followed by UMAP-based cell dimensionality reduction clustering analysis, YG021_T: tumor tissue of YG021 patient; YG021_C: conditionally reprogrammed PDC of YG021 patient; YG021_SC: stem cells derived from YG021 patient.

[0072] like Figure 7As shown in Figure 2, whole exome sequencing (WES) was performed on samples from six glioma patients, and the samples carried common glioma mutations such as TP53, PTEN, IDH1, EGFR, PIK3CA, and ATRX (such as Figure 7 (shown in A);

[0073] Two of the six patients had samples carrying glioma IDH1 mutations, including one sample with a typical IDH R132H mutation. Studies have shown that cells carrying this mutation are difficult to culture in vitro. Further Western blotting detection of these six PDCs using IDH1 R132H-specific antibodies revealed that the PDCs retained this mutation feature. The cell reprogramming method can successfully culture primary glioma cells that are difficult to construct, and enable the cells to retain the patient's genetic variation characteristics (such as Figure 7 B), where Figure 7 A in the middle shows six tumor samples that underwent WES sequencing and cancer driver gene analysis. Figure 7 B shows the Western detection of R132H mutation in PDC sample.

[0074] Embodiment 5, as Figure 8 As shown, three consecutive numbered PDC orthotopic glioma mice (PDC_DX) were constructed. PDC can stably form tumors in NCG mice. Human-derived PDC YG014_C, YG016_C and YG017_C were specifically marked by fluorescent staining of human-specific nuclear antigen HuNu. By observing the tumor growth of the entire brain slice, the tumor migration of YG016_DX mice was strong. Tumor cells were seen migrating from the tumor implantation site in the right brain of the mouse to the contralateral brain region. Tumor cells diffusely grew almost throughout the right brain, causing right brain enlargement and ventricle compression and deformation. The tumor of YG017_DX mice showed obvious invasive growth.

[0075] H&E staining of PDC_DX (eg Figure 8As shown in the figure, it can be observed that the nuclei of tumor cells in YG014_DX are darker and heteromorphic, the tumor cells are closely arranged, and the cell density is high; a large range of tumor cells are necrotic, and the nuclei are fragmented or dissolved; the tumor has multiple small focal hemorrhages and red blood cell exudates; the nuclei of tumor cells in YG016_DX are heteromorphic, mostly irregular, and occasionally nuclear division phases are seen; the boundary between tumor tissue and brain tissue is unclear, the brain tissue of YG016_DX has neuronal necrosis, nuclear dissolution, and increased cytoplasmic eosinophilia; the tumor cells in YG017_DX mice The nuclei were mostly oval, long spindle or irregular, with slightly basophilic cytoplasm; the cells were arranged in a staggered manner, and the stroma was relatively loose; the boundary between tumor tissue and brain tissue was unclear, and a small number of round vacuoles were seen in the stroma. The three PDC_DX models all had typical diffuse glioma characteristics and also showed heterogeneity of tumor growth phenotype. The cells YG014_C, YG016_C, and YG017_C from three patients were used to implant tumors in situ in the brain of NCG mice, and the human specific nuclear antigen HuNu fluorescence detection of mouse brain tissue (such as Figure 8 left) and H&E staining of mouse brain tissue sections (as shown in Figure 8 shown on the right in the figure).

[0076] In summary, a non-contact irradiation-induced conditional reprogramming method was used to construct a glioma PDC model, and for the first time, the similarity between the cultured PDC and the parental tumor was characterized at the transcriptome, genome and single-cell levels. The cultured PDC can form tumors with patient characteristics in the mouse skull. This method has the following advantages: First, the conditioned medium produced by irradiation-induced feeder cells can quickly reprogram glioma PDC in a short period of time and give it the ability to proliferate indefinitely. Second, non-contact reprogramming avoids the problem of residual feeder cells during mixed culture, improves the purity of tumor culture, and makes cell passaging and other operations simpler. Third, this method can retain all tumor subclones in the tissue, rather than culturing tumor clones with growth advantages in a competitive manner of clonal selection. Fourth, the method also has a high culture success rate for IDH1 mutant gliomas, which is the highest success rate among all current culture methods.

[0077] The established glioma PDC model replicated the key characteristics of glioma in both in vivo and in vitro experiments, and can be used for high-throughput drug screening for personalized treatment of glioma patients. It has broad application potential in basic and translational research on glioma.

[0078] Obviously, the above embodiments are only examples for clear explanation, and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A method for culturing primary cells (PDC) derived from glioma patients, characterized in that: The specific steps are as follows: S1, Swiss3T3-J2 cell culture: S11 and Swiss3T3-J2 mouse fibroblasts were cultured in DMEM complete medium and cell passage was performed after the cell confluence rate reached 60%-70%; When subculturing S12 and J2 cells, the culture medium was discarded, the adherent cells were washed once with PBS, and digested with trypsin / EDTA solution; S13. Observe the cell digestion status under a microscope. When the cells become round and begin to fall off from the bottom of the culture dish, gently tap the culture bottle to promote cell shedding. S14. Stop the trypsin / EDTA action by adding four volumes of DMEM complete medium to the cells; S15, centrifuge at 300g for 5 minutes at 4°C, resuspend the cells in 10 mL of DMEM complete medium, and inoculate 3 × 10 cells per square centimeter. 3 Cells, passaged in about three days; S2. Conditioned medium preparation: S21, Swiss3T3-J2 cells were suspended in DMEM complete medium, and irradiated with 6MV electron beam, X-ray and cobalt-60 radiation sources at radiation doses of 10 Gy, 30 Gy and 60 Gy, respectively; S22, after irradiation, 1.0×10 7 to 1.5×10 7 The cells were seeded at 175 cm 2 In the tissue culture flask, add 30 mL of F medium; S23, 72 h later, the culture medium produced by 3T3 cells was collected and centrifuged at 1000 g for 5 min at 4 °C; S24, filtering the culture medium with a filter, freezing the treated culture medium at -80°C, and before use, mixing three portions of the culture medium produced by 3T3 cells with one portion of fresh F culture medium, and adding Y-27632 (10 μM) to prepare conditioned medium CM; S3. Pretreatment of brain tumor tissue: S31. Within 1 hour after surgery, quickly rinse the excised tissue sample with 4°C precooled PBS, transfer the tissue to a sterile culture dish, remove the remaining fat tissue with forceps and a scalpel, and cut into approximately 1 cm 3 The tumor was cut out as completely as possible from the surrounding normal tissue, and at least one section of each sample was used for pathological examination; S32, place the sample in F medium (containing 10 μM Y-27632) and transport at 4°C; S4. Digestion and culture of primary tumor cells: S41, dilute 10× collagenase / hyaluronidase solution to 1× with F medium, mix 3 mL of F medium / collagenase / hyaluronidase with 1 mL of dispase; S42. Rapidly rinse the excised tissue sample with ethanol and then rinse with ice-cold PBS; S43, take 1cm 3 Use a scalpel to mince the tissue. S44, transfer the tissue fragments to a tube containing 4 mL of L F medium / collagenase / hyaluronidase / dispase and incubate on a shaker for digestion; S45. After the tissue is fully enzymatically separated, centrifuge at 500 g for 5 min at 4°C, discard the supernatant, and resuspend the cells in 10 mL of F medium; S46. Filter the cell suspension through a 100 μm cell sieve into a new 50 mL centrifuge tube; S47. Divide the cell suspension equally according to the type of culture medium and add it to the centrifuge tube. Centrifuge at 300g for 5 minutes at 4°C. Discard the supernatant to collect the cell pellet. Resuspend the cells with CM conditioned medium and then plant the cells in a T75 cell culture flask.

2. The method for culturing primary cells (PDC) derived from glioma patients according to claim 1, characterized in that: The J2 cells in S11 are prone to growth inhibition, and the growth density during subculture should not exceed 4×10 4 / cm 2 .

3. The method for culturing primary cells (PDC) derived from glioma patients according to claim 1, characterized in that: The condition for washing the adherent cells once with PBS in S12 is digestion with 0.05% trypsin / EDTA solution at room temperature for 1 minute.

4. The method for culturing primary cells (PDC) derived from glioma patients according to claim 1, characterized in that: The F medium formula in the S22 (volume or final concentration in brackets): DMEM complete medium (373 mL), F12 nutrient mix (125 mL), Hydrocortisone (25 ng / mL), EGF (0.125 ng / mL), Insulin (5 μg / mL), cholera toxin (0.1 nM), Amphotericin B (250 ng / mL), gentamicin (10 mg / mL).

5. The method for culturing primary cells (PDC) derived from glioma patients according to claim 1, characterized in that: The filter in S24 is a Millex-GP filter with a pore size of 0.22 μm.

6. The method for culturing primary cells (PDC) derived from glioma patients according to claim 1, characterized in that: The concentration of ethanol in the S42 is 95%-100%, and the flushing time is at most 3 seconds.

7. The method for culturing primary cells (PDC) derived from glioma patients according to claim 1, characterized in that: The incubation digestion temperature on the shaker in the S44 is 37°C and the time is 1-3 hours.

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